The predominant probe type in the qPCR market is linear hydrolysis probes for use in the 5′ nuclease assay. They are traditionally labeled with two dyes, generally located at the termini of a nucleic acid oligomer, for example, a 5′ fluorescent reporter and 3′ quencher, e.g., a dark quencher. The 3′ modification can serve the dual purpose of prohibiting polymerase extension from the probe upon hybridization; and quenching signal from the fluorophore before the probe hybridizes to its target sequence.
The quencher and fluorophore may interact through either Förster Resonance Energy Transfer (FRET) and/or static quenching, which it is hypothesized that they contact one another to form a ground-state complex that is non-fluorescent (Marras, S. A. E., Kramer, F. R., and Tyagi, S. (2002) Nucleic Acids Res. 30, e122; Johansson, M. K. (2006). Methods Mol. Biol. 335, 17-29). This interaction suppresses the signal when the probe is free in solution. Upon oligo hybridization, the duplex formed is quite rigid and so increases the effective length compared to single-stranded oligo, which is much more flexible. Binding of the probe to the target sequence is therefore sufficient to disrupt the interaction between dyes positioned at opposite ends of an oligo and release fluorescent signal (Parkhurst, K. M., and Parkhurst, L. J. (1995). Biochemistry 34, 285-292).
The signal release upon hybridization can be made permanent by hydrolyzing the oligo between the fluorophore and the quencher. In fact, cleavage is accomplished when Taq polymerase extends from a primer and encounters the probe in its path (FIG. 1). The nuclease activity of the polymerase cleaves the probe one or several bases from its 5′ end until the probe loses binding stability and is displaced from the strand. This hydrolysis is the basis for the signaling mechanism in TaqMan® probes.
FIG. 1 is an illustration of a 5′ nuclease (TaqMan®) assay. Briefly, after denaturation, the probe and primers anneal to the dissociated strands. During elongation the polymerase extends from the primer, encounters the probe, and hydrolyzes nucleotide fragments that are displaced from the strand. Cleavage of the probe separates the fluorophore and quencher rendering signal release permanent.
Probe performance can be quantified in a general sense by a signal-to-noise ratio: the final fluorescence following amplification divided by the initial fluorescence preceding amplification. The initial fluorescence represents the quenching efficiency, and so to double this background signal reduces the S:N by a factor of two. Quenching efficiency depends, in part, upon the oligo length. This is because FRET quenching diminishes quite rapidly with increasing separation between the dyes according to a relationship of (1/r){circumflex over ( )}6, where r is the distance through space (Cardullo et al. (1988). Proc. Natl. Acad. Sci. U.S.A. 85, 8790-8794.). Single-stranded oligos are thought to behave as a random coil and so the effective distance is the average of many conformations, but the principle remains the same: increasing sequence length diminishes the quenching efficiency, resulting in probes with elevated baseline fluorescence and poor signal-to-noise values. For this reason, probe designs are typically limited to 30 bases or shorter to achieve sufficient quenching efficiency for the final application.
Oligonucleic acid lengths are selected with consideration not only to quenching efficiency but also their binding stability. A common design guideline is to select probe sequences with a TM of 70° C., elevated above that of the primers. Probes must typically be longer than 20 bases to accomplish that TM depending on the base composition, and in the absence of special modifications to promote hybridization. Sequence design is thus a careful compromise between binding stability and quenching efficiency, but that 20-30 base window is inadequate for many difficult targets. For example, SNP genotyping requires probes shorter than 20 bases in order to achieve the enhanced specificity needed for mismatch discrimination. At such a low TM SNP probes would be nonfunctional without the use of chemical moieties to increase their binding stability—a Minor Groove Binder (MGB) (Kutyavin et al. (2000). Nucleic Acids Res. 28, 655-661) or the propynyl residues used in BHQplus probes, among others. These chemical modifications serve to relax the lower limitation, allowing the design of compact sequences beneath 20 bases in length.
Many target genes are particularly AT-rich and require longer sequences to obtain the proper TM. The upper limitation on sequence length can be relaxed by positioning the quencher at an internal location closer to the fluorescent reporter, rather than at the 3′ terminus. In fact, some of the earliest TaqMan probe designs had the quencher at an internal location for this reason—to improve their quenching efficiency (Lee et al. (1993) Nucleic Acids Res. 21(16): 3761-3766). However, the 3′ position now vacated must still be modified with a blocker such as a terminal phosphate or aliphatic carbon spacer, to prevent the probe from behaving as a primer and triggering extension.
Probes with an internal quencher do improve the quenching efficiency for sequences longer than 30-bases. Internal quenchers are traditionally positioned off of a thymidine base to preserve the sugar-phosphate backbone and presumably minimize any disruption to base-pairing. For certain assays, however, this strategy will diminish the magnitude of signal release upon amplification. The reasons for this suppression are not entirely clear, but the outcome effectively reduces the numerator of the S:N ratio, compromising probe performance. FIGS. 2A-C show representative results when Black Hole Quencher-1 (BHQ1) is positioned upon an internal T residue, across three different probe lengths.
FIGS. 2A-C. Amplification traces signaled with an end-labeled probe are shown as slashed lines. Amplification traces signaled with an internal T-BHQ1 probe are shown as solid lines. The improvement in quenching efficiency is most pronounced with longer probe sequences, while the short 21-base probe has a reduced magnitude of signal release compared to the end-labeled probe.
Tethering the BHQ1 off a thymidine constrains probe design to those sequences that have a T toward the 5′ end, since the reactive precursor used to incorporate the quencher during oligo synthesis involves a T-linked phosphoramidite. This base dependence is a significant limitation, as is the reduced signal release from select assays, particularly those with shorter probe sequences. The design of qPCR assays could be made more facile, and their signal amplification more robust by:                1. Improving both the magnitude of signal release and the quenching efficiency.        2. Eliminating any base dependencies that restrict the quencher position.        3. A label orientation that permits design of both short and long sequences with similar performance improvement.        
The present invention provides nucleic acid probes meeting these three and having other advantages as well.